New Targets and Potential Strategy to Enhance the Anticancer Efficacy BKM-120 and BEZ235 in Lymphoma Cell Lines

Background. The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays an important role in many physiological functions, including cell cycle progression, differentiation, survival, motility, apoptosis, protein synthesis and metabolism modification. The PI3K/AKT/mTOR signaling pathway also inhibits cell autophagy, a catabolic process involving the degradation of a cell’s own components through the lysosomal machinery. BKM120 and BEZ235 are synthetic small molecules belonging to the class of imidazo-quinolones that show preclinical activity against a range of solid and hematological malignancies. BKM-120 inhibits the catalytic subunit of class I PI3K by competitive binding to its ATP binding site, while BEZ235 is a dual class I PI3K/mTOR inhibitor. In this study we investigated the effects of BKM120 and BEZ235 on survival rate, apoptosis, signaling pathways expression, autophagy, metabolism modification, cell cycle arrest and tubulin polymerization kinetic in lymphoma cell lines.

Method. Lymphoma cell lines (WSU-NHL, Jeko-1 and Karpas-299) were treated with different concentrations of BKM120 and BEZ235 (Novartis) for 24 and 48h and the IC₅₀ values were evaluated using MTT assay. To assess for apoptosis, we used annexin V/PI staining kit/ flow cytometer analysis and Western Blot to evaluate caspase 3, 8, 9 and PARP expression. The cell cycle was performed applying PI incorporation and flow cytometer analysis. A flow cytometry based technique for the analysis of tubulin polymerization using a-tubulin staining was done to test whether the two drugs could cause a mitotic block. Western blot was utilized for phosphorylation status of protein kinases and for monitoring autophagy and metabolism.

Results and conclusions. BKM120 and BEZ235 induced significant increase of apoptosis evidenced by annexin IV/PI staining and confirmed by the cleavage of caspases -3, -9 -8 and PARP. BKM120 and BEZ235 induced an up regulation of pro apoptotic protein Bim, Bax and Bad. Treatment for 24h with BKM120 and BEZ235 resulted in different effects on cell cycle. BKM120 induced an increase of G2-phase with down regulation of Cyclin D and E, and an up-regulation of Cyclin A, p21 and p27. The increase in G2-M caused by BKM120 treatment occurred in a dose dependent manner. BEZ235 induced an increase of G0/G1-phase with up regulation of Cyclin A, D, E and p21 and p27. Quantitative analysis of a-tubulins polymerization of the cell lines revealed that treatment with BKM120 induced an accumulation of mitotic cells. BKM120 and BEZ235 are inhibitors of intracellular pathways in targeting p-Akt, p- mTOR, pS6K, 4EBP1, MYC and STAT. BKM120 and BEZ235 increased the levels of type II LC3 and p62, hallmarks of autophagy, in addition to increasing caspase 3 cleavage and annexin positive cells, suggesting that the two drugs induced both apoptosis and autophagy. The combination of BKM120 and BEZ235 with chloroquine which are in clinical use, with the lysosomotropic autophagy inhibitor chloroquine demonstrating marked cooperates with inhibition of autophagy to elicit apoptosis through the intrinsic mitochondrial pathway (BAX, BIM and BAD). Treatment with BKM120 and BEZ235 induced an increase of GLUT1 and HIF-1 protein expression. GLUT1 plays a role in regulation of ROS levels in particular after BEZ235 treatment. We concluded that BKM120 and BEZ235 inhibit PI3K/AKT and mTOR signaling, induce autophagy-apoptosis, affect lymphoid cell metabolism and promote ER stress via ROS increase. Moreover, we observed that BKM120 can act as a microtubule destabilizer inducing cell cycle arrest. In these study, we highlight new targets of BKM120 and BEZ235 in addition to the known PI3K/AKT signaling pathway; these results can help to identify new potential strategies to enhance the anticancer efficacy BKM-120 and BEZ235 in lymphoma cell lines.